There are a large number of methods for labeling nucleotides, oligonucleotides or nucleic acids (herein referred to by the term polynucleotides). Polynucleotides can be labeled either during synthesis (e.g., by incorporating at least one labeled nucleotide) or by adding a label to the polynucleotide after synthesis. For example, one method attaches the label to the base, whether the latter is a natural base or a modified base. A second method attaches the label to the sugar, again whether it is a natural sugar or a modified sugar. A third method attaches the label to the phosphate. Often, preferred methods attach the label to the base or to the sugar, because such methods are more convenient and provide more options for labeling. See, for example, the methods disclosed in EP-A-0.329.198, EP-A-0.302.175, EP-A-0.097.373, EP-A-0.063.879, U.S. Pat. No. 5,449,767, U.S. Pat. No. 5,328,824, WO-A-93/16094, DE-A-3.910.151 and EP-A-0.567.841 in the case of base labeling, or EP-A-0.286.898 in the case of sugar labeling. Attaching the label to the phosphate is more complex because nucleic acids are water soluble and the reactivity of the phosphate in an aqueous solution is low. Nonetheless, phosphate labeling methods have been described in EP-A-0.280.058. In this method, the label is attached to the phosphate, which is attached to the sugar in the 3′ and/or 5′ positions, for a deoxyribonucleotide, and in the 2′, 3′ and/or 5′ positions for a ribonucleotide. The labeled nucleotide may be incorporated into the polynucleotide or oligonucleotide during synthesis.
However, the labeling described in EP-A-0.280.058 does not uniformly label the nucleic acids. The incorporation of the labeled nucleotides into the polynucleotides cannot be controlled and depends on the composition of synthesized polynucleotides. Thus, some polynucleotides may contain a large number of labeled nucleotides whereas others may not contain any. As a result, the intensity of the signal emitted by these labeled nucleic acids will not be uniform, making it difficult to interpret the results when detecting the nucleic acids.
Another method, described in U.S. Pat. No. 5,317,098 relates to nucleic acids (e.g., 15-mers) which are labeled at their 5′ ends by using imidazole and a linker arm. Furthermore, phosphate is added to nucleic acids by using a kinase, thus adding at least one additional step. When this method is used to label larger nucleic acids, the specific activity is low because this technique labels only the 5′ end.
In some instances, fragmentation of a labeled nucleic acid is also desirable, such as to increase hybridization kinetics of the labeled fragment with another nucleic acid by decreasing the size of the labeled polynucleotide. In contrast, hybridization using a larger labeled polynucleotide may result in a quantitative and qualitative loss of the signal. Fragmentation of a labeled polynucleotide may also be needed to reduce steric hindrance.
Steric hindrance may result from the length of the nucleic acid and the existence of secondary structures. Fragmentation helps to remove these structures and, thus, optimize hybridization. Steric hindrance plays a particularly important role in hybridization to surfaces which contain a high density of capture probes, for example, in high-density arrays of probes as occur on “DNA chips” (GENECHIP®; Affymetrix, Santa Clara, Calif., USA; (Chee et al., 1996, Science 274:610-614; Caviani Pease et al., 1994, Proc. Natl. Acad. Sci. USA 91:5022-5026; U.S. Pat. No. 5,445,934; U.S. Pat. No. 5,744,305; Ramsay, 1998, Nature Biotechnol. 16:40-44, Ginot, 1997, Human Mutation 10:1-10; Cheng et al., 1996, Molec. Diagnosis 1(3).183-200; Livache et al., 1994, Nucl. Acids Res. 22(15): 2915-2921; Cheng et al., 1998, Nature Biotechnol. 16. 541-546; U.S. Pat. No. 5,525,464, U.S. Pat. No. 5,202,231, U.S. Pat. No. 5,807,522 and U.S. Pat. No. 5,700,637).
Methods for fragmenting nucleic acids are known in the art. For example, fragmentation can be enzymatic (i.e. by nucleases such as DNases or RNases). This generates small fragments having 3′-OH, 5′-OH, 3′-phosphate and 5′-phosphate ends. Alternatively, fragmentation can be chemical. For example, for DNA, it is possible to depurinate or depyrimidinate the DNA, which are then fragmented in the presence of a base (i.e., “β-elimination”) DNA can be fragmented by oxidation, alkylation or free radical addition mechanisms. Metal cations, which are often combined with organic molecules which may function as chemical catalysts, for example imidazole, are used for fragmenting RNA. This fragmentation is preferably carried out in an alkaline medium and generates fragments having 3′-phosphate ends.
Different nucleic acid fragmentation techniques have been described in Trawick et al., 1998, Chem Rev. 98; 939-960 and Oivanen at al., 1998, Chem Rev. 98: 961-990.
A method for fragmenting and labeling RNA is described in WO-A-88/04300, in which the fragmentation is carried out using RNA which possesses enzymatic properties (ribozymes). Fragmentation by ribozymes releases a nucleic acid (5′) HO end and a nucleic acid (3′) HO-PO2 end. Radioactive labeling is then effected by incorporating a radioactive phosphate, derived from GTP, at the 5′OH end; no phosphate resulting from fragmentation is used in labeling. Fragmentation carried out by ribozymes implies specificity between the ribozymes and the target nucleic acids to be cleaved, after which the phosphate acts as the label.
Reliable diagnostic tests based on nucleic acid amplification techniques often include steps to control contamination by nucleic acids that can otherwise serve as targets for further amplification. Several decontamination procedures have been developed (Longo et al., 1990, Gen. 93: 125-128; Abravaya et al., in Nucleic Acid Amplification Technologies, p 125-133, (1997) Eds. Lee et al. (Eaton Publishing 1997) at pp. 125-133; EP 0 709 468 Al and U.S. Pat. No. 5,605,796). These procedures make the amplified nucleic acid product incapable of being a target for further amplification, generally by degrading nucleic acids that would otherwise serve as targets (e.g., by using irradiation, endonucleases, uracil DNA glycosylase, primer modification or photochemical methods). Some of these methods are difficult to implement, are inefficient or introduce additional steps and/or toxic compounds into a procedure (e.g., UV inactivation, photochemical degradation, primer modification). Enzymatic methods use enzymes that are often expensive and incompatible with amplification and/or detection buffers. Thus, there remains a need for efficient and convenient methods of target nucleic acid removal.